The long term objectives are to define the roles of different protein assemblies in nucleotide excision repair (NER) in the eukaryote, Saccharomyces cerevisiae, to determine the sequence of events that underlie dual incision of UV damaged DNA, and to examine the role of protein phosphorylation in the modulation of activities of NER proteins and of subunits of Pol II transcriptional factor IIH (TFIIH). The proposed studies will identify the protein factors that modulate the proficiency of the reconstituted incision reaction, and will examine the biochemical properties of NER proteins individually and in protein assemblies. The Rad7 and Rad16 proteins will be purified and biochemical activities of Rad7, Rad16, and of Rad7-Rad16 complex defined. Interaction domains in Rad7 and Rad16 proteins will be identified and the role of ATP in Rad7- Rad16 complex formation examined. Whether Rad7, Rad16, and Rad7-Rad 16 proteins have affinity for UV damaged DNA will be determined. The role of PHR1-encoded photolyase in NER will be studied by genetic and biochemical means. Whether addition of the PHR1, Rad 7, and Rad16 proteins to the reconstituted system increases the efficiency of dual incision of a linear DNA fragment containing a site specific cyclobutane pyrimidine dimer (CPD) will be examined. The patterns of interactions among proteins in the Rad14-Rad1-Rad10 complex will be analyzed and DNA damage binding affinity of the complex determined. The involvement of Rad23 ubiquitin domain in Rad4-Rad23 complex formation will be examined, and the role of Rad4-Rad23 complex in assembling the NER machinery studied. The hierarchy by which different NER factors are assembled onto the DNA damage site will be analyzed by native gel electrophoresis DNA binding assay and by footprinting studies. The MMS19-associated protein complex will be purified, genes encoding MMS19-associated proteins will be cloned, and their role in NER and Pol II transcription determined. The role of MMS19 associated kinase in phosphorylation of NER proteins and of TFIIH components will be examined. Xeroderma pigmentosum (XP) patients are defective in NER of UV damaged DNA, and as a consequence, they suffer from a high incidence of skin cancers. Because of the remarkable evolutionary conservation of the NER machinery between yeast and humans, these studies should continue to provide insights into the mechanisms of this complex process in humans, and they are expected to reveal the roles of human XP genes in processes other than NER.